Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris

The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265 U/L and 300 mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145 U/L and 200 mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100 U/L) and protein yield (2.0 g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast.     

Improving the synthesis of phenolic polymer using Coprinus cinereus peroxidase mutant Phe230Ala

The F230A mutant of Coprinus cinereus peroxidase (CiP), which has a high stability against radical-inactivation, was previously reported. In the present study, the radical-robust F230A mutant was applied to the oxidative polymerization of phenol. The F230A mutant exhibited better polymerization activities than the wild-type CiP in the presence of water-miscible alcohols i.e., methanol, ethanol, and isopropanol despite its lower stability against alcohols. In particular, the F230A mutant showed a higher consumption of phenol (40%) and yielded phenolic polymer of larger molecular weight (8850 Da) in a 50% (v/v) isopropanol-buffer mixture compared with the wild-type CiP (2% and 1519 Da, respectively). In addition, the wild-type CiP and F230A mutant had no significant differences in enzyme inactivation by physical adsorption on the polymeric products or by heat incubation, and showed comparable kinetic parameters. These results indicate that high radical stability of the F230A mutant and improved solubility of phenolic polymers in alcohol-water cosolvent systems may synergistically contribute to the production of the high molecular weight phenolic polymer.     

Combined strategies for improving the heterologous expression of an alkaline lipase from Acinetobacter radioresistens CMC-1 in Pichia pastoris

In this study, a series of strategies was developed to enhance the expression of an alkaline lipase from Acinetobacter radioresistens (ARL) in Pichia pastoris. Activity of the lipase from recombinant strain carrying a single copy of codon-optimized ARL gene was 65 U/mL in shake flask culture with p-nitrophenyl caprylate as the substrate. The lipase yield was increased to 104 U/mL by introducing a short N-extension spacer peptide coding for the 10 amino acids (EEAEAEAEPK) between α-factor signal peptide and ARL. The N-terminal extension spacer did not affect the pH or temperature properties of the recombinant ARL. After the multi-copy constructs were identified by Q-PCR assay, a higher lipase activity of 180 U/mL was obtained. Further introduction of the spliced HAC1 gene into multi-copy integrants (>6 copies) extensively enhanced the ARL yield by 30–40%. As a result, the ARL yield reached 1.06 × 10⁴ U/mL in a 10-L scaled-up fed-batch fermenter as well as the lipase showed some better properties compared to that wild one from A. radioresistens.     

Expression and characterization of antimicrobial peptide CecropinAD in the methylotrophic yeast Pichia pastoris

Hybrid antibacterial peptide CecropinAD (CAD) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach to express the hybrid peptide CAD in the methylotrophic yeast Pichia pastoris, the cDNA sequence encoding CAD was obtained by recursive PCR (rPCR) and cloned into the vector pPICZα-A. The Sac I-linearized recombinant plasmid pPICZα-CAD was transformed into P. pastoris GS115 by electroporation. Expression of recombinant CAD was induced for 96 h with 1.0% methanol at 28 °C, pH 5.0. The recombinant CAD was purified by two steps of reversed-phase HPLC and 1.8 mg pure active CAD was obtained from 100 ml culture. Tricine-SDS-PAGE and mass spectrometry analyses demonstrated that the molecular weight of the purified CAD was 3.8 kDa. Analysis of circular dichroism (CD) revealed that CAD mainly has α-helixes in the presence of 10 mM phosphate buffer (pH 7.2), 50% TFE/water solution (pH 2.0), or 30 mM SDS (pH 10.8). FACScan analysis showed that the antibacterial mechanism of CAD is to act on the cell membrane to disrupt bacterial cell structure. Antimicrobial assays demonstrated that recombinant CAD has a broad spectrum of anti-microbial property against fungi, as well as Gram-positive and Gram-negative bacteria, but does not have hemolytic activity against human erythrocytes. Our results suggest that recombinant antimicrobial peptide CAD may serve as an attractive candidate for the development of therapeutic antimicrobial drugs.     

Gene cloning,overexpression and characterization of a novel organic solvent tolerant and thermostable lipase from Galactomyces geotrichum Y05

Although the lipase of Geotrichum candidum has been extensively reported, little attention has been focused on molecular genetic and biochemical characterizations of Galactomyces geotrichum lipases. A lipase gene from G. geotrichum Y05 was cloned from both genomic DNA and cDNA sources. Nucleotide sequencing revealed that the ggl gene has an ORF of 1692 bp without any introns, encoding a protein of 563 amino acid residues, including a potential signal sequence of 19 amino acid residues. The amino acid sequence of this lipase showed 86% identity to lipase of Trichosporon fermentans WU-C12. The mature lipase gene was subcloned into pPIC9K vector, and overexpressed in methylotrophic Pichia pastoris GS115. Active lipase was accumulated to the level of 100.0 U/ml (0.4 mg/ml) in the shake-flask culture, 10.4-fold higher than the activity of the original strain (9.6 U/ml). This yield dramatically exceeds that previously reported with 23–50 U/ml, 0.06 mg/ml and 0.2 mg/ml. The purified lipase exhibited several properties of significant industrial importance, such as pH and temperature stability, wide organic solvent tolerance and broad hydrolysis on vegetable oils. Such a combination of properties makes it a promising candidate for its application in non-aqueous biocatalysis, such as biodiesel production, selective hydrolysis or esterification for enrichment of PUFAs and oil-contaminated biodegradation, which have been drawn considerable attention currently.     

Optimal conditions for enhanced β-mannanase production by recombinant Aspergillus sojae

Optimization of the growth conditions for maximum β-mannanase production in shake flasks by using recombinant Aspergillus sojae ATCC11906 (AsT1) was carried out by Box–Behnken design of response surface methodology. The highest β-mannanase activity on the fourth day of cultivation at 30 °C was obtained as 363 U/ml in the optimized medium consisting of 7% sugar beet molasses, 0.43% NH₄NO₃, 0.1% K₂HPO₄ and 0.05% MgSO₄ (by weight per volume) at 207 rpm. On the sixth day of cultivation under the optimized conditions, the highest β-mannanase activity was achieved as 482 U/ml which is 1.4-fold of 352 U/ml activity found on glucose medium previously.     

Peroxidase-catalyzed copolymerization of syringaldehyde and bisphenol A

Syringaldehyde, one of the major derivatives of lignin, was copolymerized with bisphenol A via a CiP (Coprinus cinereus peroxidase)-catalyzed reaction. Although syringaldehyde was not polymerized to a solid polymer, the copolymer with bisphenol A was obtained as a dark brown powdery precipitate. The relatively hydrophobic solvent, 2-propanol, gave a better yield (yield = 95%) than hydrophilic solvents, such as methanol, ethanol or acetone. Characteristic signals corresponding to the aldehyde group of syringaldehyde in the copolymer were detected in the FT-IR and ¹³C NMR spectrum. The ratio of syringaldehyde incorporated into the copolymer was estimated by measuring the amount of monomers consumed (syringaldehyde and bisphenol A), which proportionally increased up to 80 mol% on increasing the initial ratio of syringaldehyde to bisphenol A. TGA (thermogravimetric analysis) showed that the thermally crosslinked copolymer (syringaldehyde:bisphenol A = 1:1, w/w) had a much higher thermal resistance to thermal degradation than poly(bisphenol A); 36% residue still remained under a nitrogen atmosphere, even over 800 °C. This implies that the copolymer of syringaldehyde and bisphenol A could be a new thermally stable material originating from renewable resources.     

Secretory expression and characterization of the recombinant myofibril-bound serine proteinase of crucian carp (Carassius auratus) in Pichia pastoris

The myofibril-bound serine proteinase (MBSP) is effective in the degradation of myofibrillar proteins, including myosin heavy chain (MHC), α-actinin, actin, and tropomyosin and was thus regarded as an important proteinase responsible for the metabolism of fish muscle in vivo. In order to better understand the characteristic differences between native MBSP and recombinant MBSP (rMBSP) and to obtain large quantity of MBSP for its application in protein science study, the crucian carp MBSP gene was cloned (669 bp) and expressed in Pichia pastoris (P. pastoris). The recombinant P. pastoris strain was cultured in shake flasks, and 66.85 mg rMBSP/L in the fermentation supernatant was obtained. SDS-polyacrylamide gel electrophoresis (PAGE) showed a main protein band with molecular weight of approximately 36 kDa. Substrate specificity analysis revealed that the rMBSP specifically cleaved substrates at the carboxyl side of lysine residue which differed from native MBSP that cleaved substrates at the carboxyl side of arginine and lysine residues. The optimum temperature and optimum pH range of the rMBSP were 55 °C and pH 7.5, respectively. Furthermore, similar to native MBSP, the rMBSP also revealed high thermostability and pH stability and is effective in degradation of myofibrillar proteins from the skeletal muscle of crucian carp.     

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

A xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100 °C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides (p < 0.05) but significantly reduced by calcium, cobalt and iron (p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production.     

Methanol vapor biofiltration coupled with continuous production of recombinant endochitinase Ech42 by Pichia Pastoris

Methanol biofiltration using methylotrophic microorganisms has been previously reported by various authors. In a previous study, a modified strain of Pichia pastoris was tested for the ability to produce endochitinase (Ech42) when coupled with methanol vapor biodegradation in batch tests. The next challenge was to validate the process in a continuous system. Thus, in the present study, a biofilter packed with perlite and inoculated with P. pastoris transformed with the plasmid pPIC-ech42 was used for methanol vapor biofiltration and the continuous production of recombinant endochitinase (Ech42) for 60 days. The maximum elimination capacity (EC) of methanol obtained was 1320 g m^?3 h^?1 at a loading rate of 1465 g m^?3 h^?1. The extracellular protein production rate in the leachate was 2360 μg h^?1 with a chitinase enzymatic activity of 123 U L^?1. The protein content on the biofilm samples was negligible, indicating the effectiveness of the overall process and of P. pastoris to excrete proteins. The carbon balance indicated that 81% of the consumed methanol was mineralized and 5.8% was incorporated into biomass. The results of this study and the economic balance underscore the promising application of linking methanol vapor biofiltration to the continuous production of recombinant proteins.     

Lowering induction temperature for enhanced production of polygalacturonate lyase in recombinant Pichia pastoris

Various effects of temperature on heterologous alkaline polygalacturonate lyase produced in recombinant Pichia pastoris were investigated. The results indicated that PGL activity could be improved significantly by decreasing the cultivation temperature. It was reached 931 U/mL with temperature lowered to 22 °C at the beginning of induction phase, which were 2.1-fold and 2.9-fold increase compared to that at 30 and 26 °C. The mechanisms behind the temperature effect on recombinant PGL production may be ascribed to poor cell viability, decrease of intracellular adenosine phosphate levels, of AOX activity but increase of extracellular proteases activities. Our study demonstrated that cultivation at lower temperatures resulted in higher cell viability, significant improvement of PGL stability and an increase intracellular AOX activity, but a lower activity of released host proteases which possibly caused the degradation of recombinant PGL. In addition, the evidence of higher intracellular adenosine phosphate levels but lower energy charge level was provided at a lower temperature induction.     

Cloning and overexpression of raw starch digesting α-amylase gene from Bacillus subtilis strain AS01a in Escherichia coli and application of the purified recombinant α-amylase (AmyBS-I) in raw starch digestion and baking industry

Considering the economic and industrial relevance of α-amylases used in food and starch industries, a raw starch digesting α-amylase gene (amyBS-I) from Bacillus subtilis strain AS01a was cloned and expressed in Escherichia coli BL21 cells. The gene also includes its signal peptide sequence (SPS) for facilitating the efficient extracellular expression of recombinant α-amylase (AmyBS-I) in correctly folded (enzymatically active) form. The native AmyBS-I consists of 659 amino acids with a molecular mass and pI of 72,387 Da and 5.8, respectively. The extracellular secretion of AmyBS-I after response surface optimization of culture conditions was found to be 7-fold higher as compared to its production under non-optimized conditions. Purified AmyBS-I demonstrated optimum activity at 70 °C and pH 6.0. It shows K_m and V_max values toward soluble starch as 2.7 mg/ml and 454 U/ml, respectively. Further, it does not require Ca²⁺ ion for its α-amylase activity/thermo-stability, which is an added advantage for its use in the starch industry. The AmyBS-I also hydrolyzed a wide variety of raw starches and produced maltose and glucose as main hydrolyzed products. The bread dough supplemented with AmyBS-I showed better amelioration of the bread quality as compared to the bread supplemented with commercial α-amylase.     

Enantioselective synthesis of ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE) from ethyl-3-oxo-3-phenylpropanoate using recombinant fatty acid synthase (FAS2) from Kluyveromyces lactis KCTC 7133 in Pichia pastoris GS115

Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%).     

Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression,purification and enzymatic characterization in Pichia pastoris

Xylanase is an important industrial enzyme. In this research, to improve the thermostability and biochemical properties of a xylanase from Aspergillus niger F19, five arginine substitutions and a disulfide bond were introduced by site-directed mutagenesis. The wild-type gene xylB and the mutant gene xylCX8 were expressed in Pichia pastoris. Compare to those of the wild-type enzyme, the optimal reaction temperature for the mutant enzyme increased from 45 °C to 50 °C, the half-life of the mutant enzyme extended from 10 min to 180 min, and the specific activity increased from 2127 U/mg to 3330 U/mg. However, the V_max and K_m of the mutant xylanase decreased. The enzyme activity in broth obtained from shake flask cultures could be induced to 1850 U/mL in 7 days, which is higher than results reported previously. Furthermore, the highest achievable enzyme activity was 7340 U/mL from 140 g/L of biomass in a 3 L fermentor used in our study.     

Overexpression of Candida rugosa lipase Lip1 via combined strategies in Pichia pastoris

In this study, combined strategies were employed to heterologously overexpress Candida rugosa lipase Lip1 (CRL1) in a Pichia pastoris system. The LIP1 gene was systematically codon-optimized and synthesized in vitro. The Lip1 activity of a recombinant strain harboring three copies of the codon-optimized LIP1 gene reached 1200 U/mL in a shake flask culture. Higher lipase activity, 1450 U/mL, was obtained using a five copy number construct. Co-expressing one copy of the ERO1p and BiP chaperones with Lip1p, the CRL1 lipase yield further reached 1758 U/mL, which was significantly higher than that achieved by expressing Lip1p alone or only co-expressing one molecular chaperone. When cultivated in a 3 L fermenter under optimal conditions, the recombinant strain GS115/87-ZA-ERO1p-BiP #7, expressing the molecular chaperones Ero1p and BiP, produced 13,490 U/mL of lipase activity at 130 h, which was greater than the 11,400 U/mL of activity for the recombinant strain GS115/pAO815-α-mCRL1 #87, which did not express a molecular chaperone. This study indicates that a strategy of combining codon optimization with co-expression of molecular chaperones has great potential for the industrial-scale production of pure CRL1.     

Enhanced secretion of recombinant α-cyclodextrin glucosyltransferase from E. coli by medium additives

The purpose of this study was to investigate the effect of medium additives on the secretion of recombinant α-cyclodextrin glucosyltransferase (α-CGTase) into the culture media of Escherichia coli. It is found that supplementation of the E. coli culture with SDS, glycine, Ca²⁺ or Na⁺, individually, facilitated the secretion of α-CGTase. Orthogonal experiment showed that the optimal condition to achieve maximal secretion of α-CGTase was the supplementation with 0.03% SDS, 400 mM Na⁺, 0.3% glycine and 10 mM Ca²⁺ together. Under this condition, extracellular enzyme activity reached 12.89 U/ml, which is 15 times higher than that of the culture without any additives. Further analysis showed that the permeability, fluidity and phosphatidylglycerol content of the E. coli cell membrane under the optimized condition were significantly increased in comparison to those under the control condition. These might be the potential mechanisms for the increased secretion of α-CGTase from the periplasmic compartment into the culture medium.     

Constitutive expression of a novel isoamylase from Bacillus lentus in Pichia pastoris for starch processing

Isoamylase is essential to saccharifying starch by cleavage of 1,6-glucoside linkages in starch molecules. In this study, a novel isoamylase gene from Bacillus lentus JNU3 was cloned. The open reading frame of the gene was 2412 base pairs long and encoded a polypeptide of 804 amino acids with a calculated molecular mass of 90 kDa. The deduced amino acid sequence shared less than 40% homology with that of microbial isoamylase ever reported, which indicated it was a novel isoamylase. A constitutive GAP promoter was used to express the recombinant isoamylase in the yeast Pichia pastoris by continuous high cell-density fermentation to avoid the use of methanol, which resulted in 318 U/mL extracellular isoamylase activity after 72 h in a 10 L fermenter. The recombinant enzyme was purified and characterized. It had an estimated molecular mass of 90 kDa, with its optimal activity at 70 °C, pH 6.5 and was quite stable between 30 °C and 70 °C. The recombinant isoamylase proves to be superior to pullulanase as an auxiliary enzyme in maltose production from starch. Therefore it will contribute significantly to the starch debranching process.     

Identification of a β-glucosidase from the Mucor circinelloides genome by peptide pattern recognition

Mucor circinelloides produces plant cell wall degrading enzymes that allow it to grow on complex polysaccharides. Although the genome of M. circinelloides has been sequenced, only few plant cell wall degrading enzymes are annotated in this species. We applied peptide pattern recognition, which is a non-alignment based method for sequence analysis to map conserved sequences in glycoside hydrolase families. The conserved sequences were used to identify similar genes in the M. circinelloides genome. We found 12 different novel genes encoding members of the GH3, GH5, GH9, GH16, GH38, GH47 and GH125 families in M. circinelloides. One of the two GH3-encoding genes was predicted to encode a β-glucosidase (EC 3.2.1.21). We expressed this gene in Pichia pastoris KM71H and found that the purified recombinant protein had relative high β-glucosidase activity (1.73 U/mg) at pH5 and 50 °C. The K_m and V_max with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.20 mM and 2.41 U/mg, respectively. The enzyme was not inhibited by glucose and retained 84% activity at glucose concentrations up to 140 mM. Although zygomycetes are not considered to be important degraders of lignocellulosic biomass in nature, the present finding of an active β-glucosidase in M. circinelloides demonstrates that enzymes from this group of fungi have a potential for cellulose degradation.